Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Vγ9Vδ2 T cells recognize butyrophilin 2A1 and 3A1 heteromers

Nature Immunology volume 25, pages 1355–1366 (2024)Cite this article

Subjects

Abstract

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity; however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell antigen receptor (γδTCR) in complex with BTN2A1 revealed that BTN2A1 engages the side of the γδTCR, leaving the apical TCR surface bioavailable. We reveal that a second γδTCR ligand co-engages γδTCR via binding to this accessible apical surface in a BTN3A1-dependent manner. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1–BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to mediate the γδTCR interaction; indeed, locking BTN2A1 and BTN3A1 together abrogated their interaction with γδTCR, supporting a model wherein the two γδTCR ligand-binding sites depend on accessibility to cryptic BTN epitopes. Our findings reveal a new paradigm in immune activation, whereby γδTCRs sense dual epitopes on BTN complexes.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: BTN2A1 engages the side of Vγ9.
Fig. 2: BTN2A1 forms V-shaped and head-to-tail homodimers.
Fig. 3: BTN3A1 supports binding to the apical surface of the Vγ9Vδ2+ γδTCR.
Fig. 4: BTN2A1 and BTN3A1 directly associate and form heteromers.
Fig. 5: BTN3A1 IgV domain is critical for ligand-two interacting with Vγ9Vδ2+ TCR.
Fig. 6: BTN2A1 and BTN3A1 must disengage to bind Vγ9Vδ2+ TCR.
Fig. 7: The intracellular tail of BTN2A1 mediates a pAg-dependent interaction with the BTN3A1-B30.2 domain.

Similar content being viewed by others

Data availability

The crystal structures are deposited in the PDB under accession codes 8DFW, 8DFY and 8DFX. Existing structures 4HH8, 4F80, 1QSE and 4LHU used in this paper were accessed from the PDB.

References

  1. Ribot, J. C., Lopes, N. & Silva-Santos, B. γδ T cells in tissue physiology and surveillance. Nat. Rev. Immunol. 21, 221–232 (2021).

    Article  CAS  PubMed  Google Scholar 

  2. Constant, P. et al. Stimulation of human gamma delta T cells by nonpeptidic mycobacterial ligands. Science 264, 267–270 (1994).

    Article  CAS  PubMed  Google Scholar 

  3. Tanaka, Y. et al. Natural and synthetic non-peptide antigens recognized by human gamma delta T cells. Nature 375, 155–158 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Tyler, C. J., Doherty, D. G., Moser, B. & Eberl, M. Human Vγ9/Vδ2 T cells: innate adaptors of the immune system. Cell Immunol. 296, 10–21 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. Hoeres, T., Smetak, M., Pretscher, D. & Wilhelm, M. Improving the efficiency of Vγ9Vδ2 T-cell immunotherapy in cancer. Front. Immunol. 9, 800 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Di Marco Barros, R. et al. Epithelia use butyrophilin-like molecules to shape organ-specific γδ T cell compartments. Cell 167, 203–218 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Melandri, D. et al. The γδTCR combines innate immunity with adaptive immunity by utilizing spatially distinct regions for agonist selection and antigen responsiveness. Nat. Immunol. 19, 1352–1365 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Boyden, L. M. et al. Skint1, the prototype of a newly identified immunoglobulin superfamily gene cluster, positively selects epidermal γδ T cells. Nat. Genet. 40, 656–662 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Jandke, A. et al. Butyrophilin-like proteins display combinatorial diversity in selecting and maintaining signature intraepithelial γδ T cell compartments. Nat. Commun. 11, 3769 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sandstrom, A. et al. The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vγ9Vδ2 T cells. Immunity 40, 490–500 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rigau, M. et al. Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells. Science 367, eaay5516 (2020).

    Article  CAS  PubMed  Google Scholar 

  12. Karunakaran, M. M. et al. Butyrophilin-2A1 directly binds germline-encoded regions of the Vγ9Vδ2 TCR and is essential for phosphoantigen sensing. Immunity 52, 487–498.e6 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Willcox, C. R. et al. Butyrophilin-like 3 directly binds a human Vγ4+ T cell receptor using a modality distinct from clonally-restricted antigen. Immunity 51, 813–825 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Vyborova, A. et al. γ9δ2T cell diversity and the receptor interface with tumor cells. J. Clin. Invest. 130, 4637–4651 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wang, H., Fang, Z. & Morita, C. T. Vγ2Vδ2 T cell receptor recognition of prenyl pyrophosphates is dependent on all CDRs. J. Immunol. 184, 6209–6222 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Harly, C. et al. Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset. Blood 120, 2269–2279 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Palakodeti, A. et al. The molecular basis for modulation of human Vγ9Vδ2 T cell responses by CD277/butyrophilin-3 (BTN3A)-specific antibodies. J. Biol. Chem. 287, 32780–32790 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Karunakaran, M. M. et al. A distinct topology of BTN3A IgV and B30.2 domains controlled by juxtamembrane regions favors optimal human γδ T cell phosphoantigen sensing. Nat. Commun. 14, 7617 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gu, S. et al. Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on Vγ9Vδ2 T cell activation. Proc. Natl Acad. Sci. USA 114, E7311–E7320 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Vantourout, P. et al. Heteromeric interactions regulate butyrophilin (BTN) and BTN-like molecules governing γδ T cell biology. Proc. Natl Acad. Sci. USA 115, 1039–1044 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cano, C. E. et al. BTN2A1, an immune checkpoint targeting Vγ9Vδ2 T cell cytotoxicity against malignant cells. Cell Rep. 36, 109359 (2021).

    Article  CAS  PubMed  Google Scholar 

  22. Hsiao, C.-H. C., Nguyen, K., Jin, Y., Vinogradova, O. & Wiemer, A. J. Ligand-induced interactions between butyrophilin 2A1 and 3A1 internal domains in the HMBPP receptor complex. Cell Chem. Biol. 29, 985–995 (2022).

    Article  CAS  PubMed  Google Scholar 

  23. Yuan, L. et al. Phosphoantigens glue butyrophilin 3A1 and 2A1 to activate Vγ9Vδ2 T cells. Nature 621, 840–848 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wang, H. et al. Conservation of nonpeptide antigen recognition by Rhesus monkey Vγ2Vδ2 T cells. J. Immunol. 170, 3696–3706 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Ding, Y. H. et al. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Immunity 8, 403–411 (1998).

    Article  CAS  PubMed  Google Scholar 

  26. Uldrich, A. P. et al. CD1d-lipid antigen recognition by the γδ TCR. Nat. Immunol. 14, 1137–1145 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Holst, J. et al. Generation of T-cell receptor retrogenic mice. Nat. Protoc. 1, 406–417 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Le Nours, J. et al. Atypical natural killer T-cell receptor recognition of CD1d-lipid antigens. Nat. Commun. 7, 10570 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Kim, H. K. et al. Deep learning improves prediction of CRISPR-Cpf1 guide RNA activity. Nat. Biotechnol. 36, 239–241 (2018).

    Article  CAS  PubMed  Google Scholar 

  30. Moll, J. R., Ruvinov, S. B., Pastan, I. & Vinson, C. Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10−15 M. Protein Sci. 10, 649–655 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Aricescu, A. R., Lu, W. & Jones, E. Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D. Biol. Crystallogr. 62, 1243–1250 (2006).

    Article  PubMed  Google Scholar 

  32. Aragao, D. et al. MX2: a high-flux undulator microfocus beamline serving both the chemical and macromolecular crystallography communities at the Australian Synchrotron. J. Synchrotron Radiat. 25, 885–891 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D. Biol. Crystallogr. 67, 271–281 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Eichinger, A., Neumaier, I. & Skerra, A. The extracellular region of bovine milk butyrophilin exhibits closer structural similarity to human myelin oligodendrocyte glycoprotein than to immunological BTN family receptors. Biol. Chem. 402, 1187–1202 (2021).

    Article  CAS  PubMed  Google Scholar 

  35. Allison, T. J., Winter, C. C., Fournie, J. J., Bonneville, M. & Garboczi, D. N. Structure of a human gammadelta T-cell antigen receptor. Nature 411, 820–824 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D. Biol. Crystallogr. 68, 368–380 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D. Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank C. Panousis, G. Matthews and staff at CSL Limited for providing BTN2A1 monoclonal antibody, M. Hattarki from CSIRO for support with SPR, the C3 protein crystallization facility (CSIRO), the Ian Holmes Imaging Centre (Bio21 Institute, University of Melbourne), the MX beamlines at the Australian Synchrotron, the Melbourne Cytometry Platform (University of Melbourne) and AGRF. This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the ACRF detector. Infrastructure support from the NHMRC Independent Research Institutes Infrastructure Support Scheme and the Victorian State Government Operational Infrastructure Support Program are gratefully acknowledged. Funding support was provided by the Miller Foundation Research Accelerator fund (to A.P.U.); the Cancer Council of Victoria 1126866 (to A.P.U.); the National Health and Medical Research Council of Australia 1184906 (to A.P.U.), 1165467 (to A.P.U.), 1194263 (to M.W.P.), 1117766 (to D.I.G.) and 2008913 (to D.I.G.); the Australian Research Council DP230102753 (A.P.U.), CE140100011 (to D.I.G.) and DE210100705 (to N.A.G.); a Cancer Council Victoria Postdoctoral Research Fellowship (to M.R.); and the Department of Health and Human Services acting through the Victorian Cancer Agency fellowship (to A.B.).

Author information

Author notes

  1. Marc Rigau

    Present address: Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

  2. These authors jointly supervised this work: Dale I. Godfrey, Adam P. Uldrich.

Authors and Affiliations

  1. Department of Microbiology & Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia

    Thomas S. Fulford, Caroline Soliman, Rebecca G. Castle, Marc Rigau, Zheng Ruan, Rebecca Seneviratna, Shihan Li, Samuel J. Redmond, Amy Chung, Nicholas A. Gherardin, Dale I. Godfrey & Adam P. Uldrich

  2. Institute of Molecular Medicine and Experimental Immunology, Rheinische Friedrichs-Wilhelms University of Bonn, Bonn, Germany

    Marc Rigau

  3. Biomedical Manufacturing, Commonwealth Scientific and Industrial Research Organisation, Parkville, Victoria, Australia

    Olan Dolezal, Thomas S. Peat & Janet Newman

  4. Ian Holmes Imaging Centre, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia

    Hamish G. Brown & Eric Hanssen

  5. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia

    Eric Hanssen, Michael A. Gorman & Michael W. Parker

  6. ARC Industrial Transformation Training Centre for Cryo-electron Microscopy of Membrane Proteins, University of Melbourne, Parkville, Victoria, Australia

    Eric Hanssen & Michael W. Parker

  7. CSL Limited, Bio21 Molecular Science and Biotechnology Institute, Parkville, Victoria, Australia

    Andrew Hammet

  8. St. Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia

    Michael W. Parker

  9. The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Onisha Patel

  10. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia

    Onisha Patel

  11. Olivia Newton-John Cancer Research Institute, and School of Cancer Medicine, La Trobe University, Heidelberg, Victoria, Australia

    Andreas Behren

  12. Department of Medicine, University of Melbourne, Parkville, Victoria, Australia

    Andreas Behren

  13. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

    Adam P. Uldrich

Authors
  1. Thomas S. Fulford
    View author publications

    Search author on:PubMed Google Scholar

  2. Caroline Soliman
    View author publications

    Search author on:PubMed Google Scholar

  3. Rebecca G. Castle
    View author publications

    Search author on:PubMed Google Scholar

  4. Marc Rigau
    View author publications

    Search author on:PubMed Google Scholar

  5. Zheng Ruan
    View author publications

    Search author on:PubMed Google Scholar

  6. Olan Dolezal
    View author publications

    Search author on:PubMed Google Scholar

  7. Rebecca Seneviratna
    View author publications

    Search author on:PubMed Google Scholar

  8. Hamish G. Brown
    View author publications

    Search author on:PubMed Google Scholar

  9. Eric Hanssen
    View author publications

    Search author on:PubMed Google Scholar

  10. Andrew Hammet
    View author publications

    Search author on:PubMed Google Scholar

  11. Shihan Li
    View author publications

    Search author on:PubMed Google Scholar

  12. Samuel J. Redmond
    View author publications

    Search author on:PubMed Google Scholar

  13. Amy Chung
    View author publications

    Search author on:PubMed Google Scholar

  14. Michael A. Gorman
    View author publications

    Search author on:PubMed Google Scholar

  15. Michael W. Parker
    View author publications

    Search author on:PubMed Google Scholar

  16. Onisha Patel
    View author publications

    Search author on:PubMed Google Scholar

  17. Thomas S. Peat
    View author publications

    Search author on:PubMed Google Scholar

  18. Janet Newman
    View author publications

    Search author on:PubMed Google Scholar

  19. Andreas Behren
    View author publications

    Search author on:PubMed Google Scholar

  20. Nicholas A. Gherardin
    View author publications

    Search author on:PubMed Google Scholar

  21. Dale I. Godfrey
    View author publications

    Search author on:PubMed Google Scholar

  22. Adam P. Uldrich
    View author publications

    Search author on:PubMed Google Scholar

Contributions

A.P.U., D.I.G., T.S.F., N.A.G. and A.B. conceptualized the project; T.S.F., C.S., M.R., O.D., S.L., A.C., T.S.P., N.A.G. and A.P.U. developed methodology; T.S.F., C.S., R.G.C., M.R., O.D., H.G.B., E.H. and A.P.U. performed experiments; Z.R., R.S., A.H., S.L., S.J.R., M.A.G., M.W.P., O.P., J.N., A.B., N.A.G., D.I.G. and A.P.U. provided resources; A.P.U. and T.S.F. wrote the initial draft; A.P.U., D.I.G., T.S.F., N.A.G., C.S., E.H., T.S.P., M.R., M.W.P., O.P. and A.H. reviewed and edited the manuscript; A.P.U., D.I.G. and T.S.F. supervised the research; A.P.U., D.I.G. and A.B. acquired funding.

Corresponding authors

Correspondence to Dale I. Godfrey or Adam P. Uldrich.

Ethics declarations

Competing interests

A.H. is an employee of CSL Limited and can partake in employee share schemes. A.B. and A.H. are inventors on a patent regarding the use of BTN2A1 to influence immune reactions (WO2015077844). T.S.F., M.R., A.H., A.B., D.I.G. and A.P.U. are inventors on a patent regarding methods of inhibiting or activating γδ T cells (WO2020257871). A.P.U., D.I.G., T.S.F., N.A.G. and M.R. are inventors on filed patents regarding BTN-mediated activation of γδ T cells. The other authors declare no competing interests.

Peer review

Peer review information

Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: S, Houston in collaboration with the Nature Immunology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Structure of BTN2A1 in complex with Vγ9Vδ2+ TCR.

(a) Unbiased 2mFo-DFc electron density composite omit map contoured at 1σ, blue mesh, surrounding γδTCR-BTN2A1 complex. (b) Size exclusion (S200 16/600) gel filtration chromatogram of BTN2A1 (black) and BTN3A1 (red) ectodomains produced in MGAT1-deficient Expi293F cells. Larger elution volume indicates smaller protein size. Data representative of at least three independent experiments. (c) Unbiased 2mFo-DFc electron density composite omit map contoured at 1σ, blue mesh, surrounding apo BTN2A1 (averaged over five copies of BTN2A1). (d) Overlay of BTN2A1 V-dimer structures derived from the apo (blue) and γδTCR-liganded (green) crystal structures. (e) Overlay of BTN2A1 head-to-tail dimers derived from the apo BTN2A1 (grey) and γδTCR-BTN2A1 (unliganded copy of BTN2A1, red) crystal structures.

Extended Data Fig. 2 Soluble Vγ9Vδ2+ TCR tetramers interact with BTN3A1 expressing cells.

(a) BTN2A1 tetramer, BTN3A1 tetramer, control mouse CD1d tetramer, or SAv-PE staining of human HEK293T cells transfected with plasmids co-encoding GFP and either G115 Vγ9Vδ2+ or control 9C2 Vγ5Vδ1+ γδTCRs. Plots gated on GFP+ cells. Data from one of 10 independent experiments. Inset – median fluorescence intensity (MFI) of PE parameter. (b) Vγ9Vδ2+ TCR tetramer-PE (clones TCR3, TCR6, TCR7 and G115) or streptavidin (SAv.) control staining of mouse NIH-3T3 cells transfected with BTN2A1, BTN3A1 or no DNA following pre-incubation with anti-BTN3A mAb clones 20.1 (red) or 103.2 (blue), or isotype control (IgG1,κ, black). Representative of N = 4 (TCR3, TCR6 and TCR7) or 2 (G115) experiments. (c) γδTCR tetramer or SAv control staining of HEK293T BTN2A.BTN3AKO cells transfected with plasmids co-encoding GFP and either BTN2A1, BTN3A1 or control BTNL3, which were pre-incubated with anti-BTN3A mAb 20.1 or isotype control (mouse IgG1,κ) antibody. Representative of one of two independent experiments. (d) SDS-PAGE separation of wild-type and Vδ1–chimeric γδ TCRs alone (LHS) or mixed with excess SAv (RHS). (e) Staining of BTN2A1, BTN3A1 or control BTNL3-transfected NIH-3T3 cells with chimeric γδTCR tetramers comprised of the TCR6, TCR7 or G115 pAg-reactive γ-chains, plus either the pAg-reactive Vδ2+ or the 9C2 Vδ1+ δ-chains ± anti-BTN3A mAb 20.1 (red) or isotype control (IgG1,κ, black). Inset of panels (c) and (e), MFI of PE for mAb 20.1-treated cells (red numbers) or isotype control (IgG1,κ)-treated BTN3A1+ cells (black numbers) on GFP+ gated events, as depicted on the SAv controls and applied to all samples. Representative of N = 3 (TCR6 and G115) or 5 (TCR7 or streptavidin (SAv)) experiments.

Extended Data Fig. 3 Residues in the CDR2δ of Vδ2Vγ9+ TCR mediate the interaction with BTN3A1-expressing cells.

(a) and (b) CD69-PE expression on G115 WT or mutant Vγ9Vδ2+, 9C2 Vγ5Vδ1+ γδTCR or parental (TCR–) J.RT3-T3.5 Jurkat cells after overnight co-culture with LM-MEL-75 APCs in the presence (red) or absence (blue) of 40 μM zoledronate. Graphs are presented as mean ± SEM. N = 2, where each point represents an independent experiment. Inset - surface representation of G115 Vγ9Vδ2+ TCR (γ-chain, orange; δ-chain, light pink) depicting the interacting (red) and gatekeeper (green) or uninvolved (gray) residues based on Jurkat activation assays. (c) γδTCR tetramer staining of wild-type or mutant G115 Vγ9Vδ2+ TCR tetramer staining, or control mouse CD1d-α-GalCer (mCD1d tet.) or streptavidin alone (SAv) staining of NIH-3T3 cells transfected with BTN2A1, BTN3A1 or control BTNL3 ± anti-BTN3A mAb clone 20.1 (red) or isotype control (IgG1,κ, black). Triple-γ mutant comprises Arg20γ-Ala/Glu70γ-Ala/His85γ-Ala mutations. Cartoon inset depicts the locations of BTN2A1-epitope (red star) and the ligand-two epitope (green star). Representative of one of three independent experiments. Inset, median fluorescence intensity (MFI) of PE for mAb 20.1-treated cells (red numbers) or isotype control (IgG1,κ)-treated BTN3A1+ cells (black numbers) on GFP+ gated events, as depicted on the SAv controls and applied to all samples. Representative of N = 10 (G115 WT, His85γ-Ala, Sav), 7 (Lys53δ-Ala), 6 (Lys108γ-Ala, Glu52δ-Ala), 4 (Arg51δ-Ala, His85γ/Glu52δ, mCD1d) or 3 (Arg20γ/Glu70γ/His85γ) experiments. (d) GFP+ BTN2A1-transfected or GFP+ BTN3A1-transfected NIH-3T3 cells were stained with streptavidin (SAv)-PE control, Vγ9Vδ2+ ‘G115 WT’, ‘G115 Lys53δ-Ala’, ‘TCR 6 WT’ or ‘TCR 6 Lys53δ-Ala’ TCR tetramers. Data from one of two independent experiments. (e) Mouse 3T3 fibroblasts expressing BTN2A1 or BTN3A1 were treated with anti-BTN3A antagonist mAb (103.2; orange) or an isotype control (MOPC-21; blue) and subsequently stained with control SAv-PE or Vγ9Vδ2 TCR tetramer-PE (wild-type or Lys53-Ala mutants of clones G115 and TCR6). Median fluorescence intensity (MFI) of PE for mAb 103.2-treated cells (orange numbers) or isotype control (IgG1,κ)-treated cells (blue numbers) shown within the depicted GFP+ gate. Representative one of three independent experiments. (f) GFP+ BTN2A1-transfected or GFP+ BTN3A1-transfected NIH-3T3 cells were stained with isotype control (MOPC-21)-AF647 or anti-BTN3A (20.1)-AF647 antibodies followed by control SAv-PE, Vγ9Vδ2 ‘G115 WT’ or ‘G115 Lys53δ-Ala’ TCR tetramer-PE staining. Cells were examined for FRET in the yellow-green (YG) 670/30 channel by flow cytometry. (g) Mouse 3T3 fibroblasts expressing BTN3A1, BTN3A2 or BTN3A3 were probed for binding by Vγ9Vδ2 TCR PE-tetramers ‘G115 WT’, ‘G115 Lys53δ-Ala’ or ‘G115 Glu52δ-Ala’, or SAv-PE control. Data in f and g are from one of two independent experiments.

Extended Data Fig. 4 BTN2A1 and BTN3A1 interact via their IgV domains.

Sensorgrams (left) and saturation plots (right) depicting binding of soluble homodimeric BTN3A1 ectodomain (top row, 1600–12 µM analyte concentration), monomeric BTN2A1 ectodomain (second row, 1400–11 µM), monomeric BTN3A1 IgV domain (third row, 2500–20 µM), monomeric BTN2A1 IgV domain (fourth row, 1700–14 µM), monomeric BTN1A1 ectodomain (fifth row, 1700–13 µM and 580–4.5 µM), homodimeric BTN3A1 ectodomain with Tyr105-Ala mutation (sixth row, 630–4.9 µM and 630–4.9 µM), irrelevant nanobody control (clone NbE1, SARS-CoV-2 RBD-specific, 1100–8.7 µM and 1200–9.4 µM), or control human ACE2 (bottom row, 210–1.6 µM and 150–1.2 µM) to immobilised BTN2A1 ectodomain homodimer (red) or BTN3A1 ectodomain homodimer (blue), as measured by surface plasmon resonance. KD, dissociation constant calculated at equilibrium. Data are from one experiment wherein BTN1A1, BTN3A1 Tyr105-Ala, NbE1 and ACE2 were performed in duplicate using two independently prepared batches of analyte, shown separately on saturation plots as upright and inverted triangles. BTN3A1 ectodomain, BTN2A1 ectodomain, BTN3A1 IgV domain and BTN2A1 IgV domain were performed with a single batch of each analyte. Data from the saturation plots and Scatchard plots in the top three rows (BTN3A1, BTN2A1 and BTN3A1 IgV domain) are also depicted as triangles in Fig. 4a.

Extended Data Fig. 5 Generation of soluble BTN2A1–BTN3A1 ectodomain complexes, which interact via their IgV domains.

(a) Representation of BTN2A1–BTN3A1–zipper complex. (b, c) (LHS) BTN2A1–BTN3A1 complex was expressed in Expi293F cells and purified by (B) affinity (NiNTA) and (C) size exclusion (S200) chromatography. (RHS) Protein purified in boxes run over SDS-PAGE to confirm identity. MM – molecular weight marker; 2A1 – BTN2A1–acid zipper (AZ)–His6; 3A1 – BTN3A1–basic zipper (BZ)–Biotin ligase tag. (d) Reactivity of anti-BTN2A1 (clone 259, red), anti-BTN3A (clone 103.2, orange), mouse IgG2a isotype control (clone BM4-2a, blue), or mouse IgG1 isotype control (clone MOPC-21, green) to immobilized BTN1A1, BTN2A1, BTN3A1 or BTN2A1–BTN3A1–zipper ectodomains by ELISA. (e) BTN2A1–BTN3A1–zipper complex was crystallized, resolubilized and run on SDS-PAGE, along with crystal wash buffer and input BTN2A1–BTN3A1–zipper complex. MM – molecular weight marker; 2A1 – BTN2A1–AZ–His6; 3A1 – BTN3A1–BZ–Biotin ligase tag. (f) 2mFo-DFc electron density composite omit map contoured at 1σ, blue mesh, surrounding BTN2A1 and BTN3A1 molecules. (g) Comparison of the apo BTN3A1 homodimer (PDB code 4F80, red cartoon) with BTN3A1 homodimer from the BTN2A1–BTN3A1–zipper complex (PDB code 8DFX, blue cartoon), and a comparison of apo BTN2A1 homodimer (PDB code 8DFY, orange cartoon) with BTN2A1 homodimer from the BTN2A1–BTN3A1-zipper complex (green cartoon). (h) Surface representation of BTN2A1 (green) and BTN3A1 (blue) depicting the regions that are contacting each other (red).

Extended Data Fig. 6 Residues in the CFG face of BTN3A1 IgV domain are required for interacting with BTN2A1 and Vγ9Vδ2+ TCR.

Summary of the effect of single-residue mutations within the (a) IgV domain or (b) IgC domain of BTN3A1 on anti-BTN3A reactivity (mAb clones 103.2 and 20.1) as well as binding in cis to BTN2A1 as measured by FRET, and binding to G115 γδTCR tetramer. +++ high expression; + intermediate expression; - low expression;–no expression; ND – no binding data due to lack of BTN3A1 expression or lack of 20.1 mAb binding. (c) Förster resonance energy transfer (FRET, depicted on x-axis) between anti-BTN2A1 (clone 259, not depicted) and anti-BTN3A (clone 103.2, depicted on y-axis) mAb staining on gated BTN2A1+BTN3A1+ NIH-3T3 cells, 48 h after co-transfection with WT BTN2A1 plus the indicated BTN3A1 mutant, or as irrelevant controls, BTN2A1 plus PD-L2 or BTN3A1 plus CD80. Mutants in red were excluded from analysis due to diminished BTN3A1 staining. Mutants in green are those which reduced FRET levels. Representative one of six independent experiments, except for CD80 and PD-L2 controls which were included in three experiments. (d) Surface representation of BTN3A1 V-dimer depicting residue side chains that upon mutation led to an abrogation of BTN3A1 association with BTN2A1 (green), or those which did not impact the interaction with BTN2A1 (black), as determined by the FRET assay (left). The BTN3A1 surface on the right depicts atoms that contacted BTN2A1 based on the crystal structure (green, reproduced from Extended Data Fig. 5b). (e) G115 tetramer-PE staining of BTN3A1 WT or mutant-transfected NIH-3T3 cells following pre-incubation with anti-BTN3A-AF647 (mAb clone 20.1). Mutants in red were excluded from analysis due to diminished BTN3A1 mAb 20.1 staining. Mutants in green are those which impaired G115 tetramer staining. Representative of one of three independent experiments. (f) CD25-PE expression on purified pre-expanded Vδ2+ γδ T cells following co-culture with NIH-3T3 cells that were co-transfected with BTN2A1 plus the indicated BTN3A1 mutant, or alternatively control BTNL3 plus BTNL8, ± zoledronate (5 µM) for 24 h. Data are from one of three independent experiments, each with two donors. (g) Surface of BTN2A1 V-dimer depicting residues that contact BTN3A1 based on the BTN2A1–BTN3A1 crystal structure in yellow, residues that contact Vγ9Vδ2+ TCR based on the G115 TCR–BTN2A1 crystal structure in blue, and residues that overlap and contact both in green. (h) Superimposition of BTN2A1 component within BTN2A1–G115 Vγ9Vδ2+ TCR and BTN2A1–BTN3A1 structures, showing BTN2A1 (green ribbon)–BTN3A1 (blue mesh) and BTN2A1 (not depicted)–G115 Vγ9Vδ2+ TCR (surface render).

Extended Data Fig. 7 Di-sulfide linked linked BTN2A1–BTN3A1 ectodomain complexes.

(a) Structure of BTN2A1–BTN3A1 depicting the locations of the two cysteine mutant pairs. (b) Predicted structure of the BTN2A1–BTN3A1 complex containing a disulphide bond between BTN2A1 and BTN3A1 molecules, based on the BTN2A1–BTN3A1–zipper ectodomain crystal structure. (c) 2D class averages of negatively stained soluble BTN2A1 Gly102-Cys–BTN3A1 Asp103-Cys ectodomain complex. Each box is 481 Å across.

Extended Data Fig. 8 Residues in the tail of BTN2A1 are required for pAg-activation of Vγ9Vδ2 T cells.

(a) Surface BTN2A1 expression (clone 259) on HEK293T BTN2AKO.BTN3AKO cells that were transfected with BTN2A1 WT or the indicated BTN2A1 intracellular domain mutants, or control BTNL3. Representative from one of two experiments. (b) Representative plots of CD25-PE expression on purified pre-expanded Vδ2+ γδ T cells following co-culture with HEK293T BTN2AKO.BTN3AKO cells that were co-transfected with BTN3A1 plus the indicated BTN2A1 mutant, or alternatively, control BTNL3 alone or BTN3A1 alone, ± zoledronate (5 µM) for 24 h. Replicates with low transfection efficiency (<10% GFP+) were excluded from analysis. Data are from three independent experiments each with n = 2 different donors.

Extended Data Fig. 9 Model of Vγ9Vδ2+ TCR interaction with BTN2A1–BTN3A1 complexes on the cell surface.

Proposed model(s) of Vγ9Vδ2+ TCR interacting with BTN2A1 and ligand two, either BTN3A or an as-of-yet unidentified third ligand, following activation of the BTN complex on APCs with activation. Created with BioRender.com.

Extended Data Fig. 10 Residues in the CDR2δ are conserved between human and primate orthologues of TRDV2.

Arg51δ, Glu52δ and Lys53δ, red box; TRDV2 alignment from ensembl.org.

Supplementary information

Supplementary Information

Supplementary Tables 1–6 and Fig. 1.

Reporting Summary

Peer Review File

Supplementary Video 1

Steric clash between Vγ9Vδ2+ TCR and BTN2A1–BTN3A1 complex. Superimposition of BTN2A1 component within BTN2A1–G115 Vγ9Vδ2+ TCR and BTN2A1–BTN3A1 structures, showing BTN2A1 (green cartoon)–BTN3A1 (blue mesh) and BTN2A1–G115 Vγ9Vδ2+ TCR (surface render).

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fulford, T.S., Soliman, C., Castle, R.G. et al. Vγ9Vδ2 T cells recognize butyrophilin 2A1 and 3A1 heteromers. Nat Immunol 25, 1355–1366 (2024). https://doi.org/10.1038/s41590-024-01892-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41590-024-01892-z

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing